Ying-Hsiu Su

Detection of a K-ras mutation in urine of patients with colorectal cancer

We previously demonstrated that human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation, which may be useful in the detection of colorectal cancer. In this report we have determined the stability of DNA in urine and have found that the half-life time interval of this small, fragmented DNA is at least 4 hours post collection. We further compared, in a blinded study, the frequency of detecting mutated K-ras sequence in DNA isolated from plasma and urine derived from individuals who have either a colorectal carcinoma (CRC), or adenomatous polyps that contain a mutation in codon 12 of the K-ras proto-oncogene. There was an 83% concurrence of mutated DNA detected in urine and its corresponding disease tissue from the same individuals, when paired urine and tissue sections from 20 patients with either CRC or adenomatous polyps were analyzed for K-ras mutation. However, only a 56% concurrence was observed when the matched plasma specimens were tested from these 20 patients. These results suggest that urine might be a better resource for detecting K-ras mutation in circulating DNA.

Rapid and sensitive detection of hepatitis B virus 1762T/1764A double mutation from hepatocellular carcinomas using LNA-mediated PCR clamping and hybridization probes

The 1762T/1764A double mutation of the hepatitis B virus (HBV) basal core promoter has been suggested to be a potential biomarker for hepatocellular carcinoma (HCC) among individuals with chronic HBV infection. In this study, a real-time PCR assay is established using the hybridization probes and an oligonucleotide clamp containing locked nucleic acids (LNAs). The LNA-containing oligonucleotide clamp specific for the wild type HBV is able to suppress the amplification of the wild type HBV templates. In addition, the clamp can inhibit the binding of the WT templates to the fluorescence probes thereby suppress the wild type HBV signals during the melting curve analyses. These effects facilitated the detection of HBV double mutation in the presence of 3000-fold excess of the wild type genome. Thus PCR amplification coupled with the melting curve analyses provides a quick, simple, and highly sensitive tool for the detection of this HBV double mutation.

Challenges for the application of DNA methylation biomarkers in molecular diagnostic testing for cancer

Aberrant DNA methylation is ubiquitous in human cancer and has been shown to occur early during carcinogenesis, thus providing attractive potential biomarkers for the early detection of cancer. The introduction of genome-wide DNA methylation analysis comparing tumor and nonmalignant tissues resulted in the discovery of many regions that undergo aberrant methylation during carcinogenesis. Those regions can potentially be used as biomarkers for cancer detection. However, a biomarker will be useful for screening or early detection of cancer only if it can be detected in a noninvasive or minimally invasive fashion without tissue biopsy. The authors discuss the challenges in translating DNA methylation biomarkers to cancer diagnosis – including obstacles in assay development, tissue-specific methylation load on tumor suppressor genes, detecting markers with sufficient sensitivity and specificity in the periphery, and ways in which these obstacles can be overcome.

DNA markers in molecular diagnostics for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the one of the leading causes of cancer mortality in the world, mainly due to the difficulty of early detection and limited therapeutic options. The implementation of HCC surveillance programs in well-defined, high-risk populations were only able to detect about 40–50% of HCC at curative stages (Barcelona Clinic Liver Cancer stages 0 & 1) due to the low sensitivities of the current screening methods. The advance of sequencing technologies has identified numerous modifications as potential candidate DNA markers for diagnosis/surveillance. Here we aim to provide an overview of the DNA alterations that result in activation of cancer pathways known to potentially drive HCC carcinogenesis and to summarize performance characteristics of each DNA marker in the periphery (blood or urine) for HCC screening.

Evidence that herpes simplex virus DNA derived from quiescently infected cells in vitro, and latently infected cells in vivo, is physically damaged

Using polymerase chain reaction (PCR) and alkaline gel electrophoresis, the authors show that, compared with DNA derived from virions used to establish infection, herpes simplex virus DNA derived from quiescently infected rat pheochromocytoma (PC12) cells in culture accumulates alkaline-labile lesions. That is, compared with equivalent amounts of virion DNA, viral DNA from nerve growth factor-differentiated long-term infected cells in culture is consistently 3 to 10 times more refractory to amplification by PCR. Despite using equal mole amounts of DNA isolated from quiescently infected cells (determined by quantitative Southern blots), DNA from quiescently infected cells could not be detected by PCR under conditions in which the virionderived DNA was easily detected. Refractoriness to PCR was confirmed by analysis with a ligation-mediated PCR technique. The refractoriness was not the result of genomic circularization. The refractoriness was, however, related to the time that the quiescently infected cells had been maintained in culture. The refractoriness to PCR was taken as an indication that the viral DNA was damaged. This hypothesis was confirmed by showing that viral DNA from quiescently infected PC12 cells accumulated alkaline-labile DNA lesions, as determined by alkaline gel electrophoresis. The phenomenon was not limited to tissue culture, because viral DNA derived from the ganglia of latently infected mice is also 3 to 10 times more refractory to amplification than are equivalent amounts of virion-derived genomes. Taken together, these results represent the first evidence that herpes simplex virus DNA is physically damaged as a function of long-term infection. Implications for viral reactivation and pathogenesis are discussed.

Abstract 876: Comprehensive analysis of the complexity of HBV DNA integration sites in the circulation of patients with HBV-related liver disease

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA HBV related liver disease ranges from hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), which has a 5-year survival rate of 14% because it is difficult to diagnose at its curative stages. Chronic hepatitis B virus (HBV) infections is the major etiology of HCC, associated with over 50% of HCC cases worldwide and up to 70-80% in endemic areas. 85-90% of HBV related HCCs (HBV-HCC) contain integrated HBV DNA. During chronic HBV infection, HBV DNA has been shown to integrate at random sites in the host chromosome creating a unique genetic signature in each HBV infected hepatocytes. This makes the pool of HBV DNA integration sites highly complex in the liver. This genetic signature in HBV-infected liver along with the fundamental mechanism of carcinogenesis, which is clonal expansion, would result in HCC tissue containing only one to few clonally expanded integration sites, resulting in reduced complexity. Thus, we hypothesize the detection of reduced complexity of HBV DNA integration sites in the circulation can be a potential biomarker for HBV-HCC. In order to study the complexity of cell free circulating HBV DNA integration sites in the circulation, a targeted next generation sequencing (NGS) assay for urine DNA was developed. We chose to use urine in order to avoid detecting of HBV DNA from infectious viruses. Targeted enrichment was performed using an in-solution hybridization platform with biotinylated RNA baits covering the entire HBV genome. The assay was tested using library constructed DNA controls: Hep3B, which contains integrated DNA, and HepG2, which contains no HBV DNA to confirm targeted capture of HBV DNA. Next, matching tissue and urine DNA and DNA from urine collected after surgical removal of HCC tumor from seven HBV-HCC patients were collected and underwent library construction. The library DNA samples were subsequently enriched with HBV and HCC-specific RNA baits. Enriched library DNA was sequenced on Illuminas MiSeq platform. Although the analysis is still ongoing, we have successfully analyzed the complexity of cell-free circulating HBV DNA integration sites in urine. We detected 46 integration sites in the tissue and urine where the location of integrated HBV DNA mapped primarily (89%) to a known HBV integration breakpoint region, HBV DR1-2. Furthermore in 3 HBV-HCC patients, a pattern of reduced complexity of cell-free circulating HBV DNA integration sites was found in urine before the removal of HCC, as compared to urine collected after surgery suggesting reduced complexity of HBV DNA integration sites in HBV-HCC patients. A similar NGS analysis of the urine of hepatitis and cirrhosis patients is in progress. Overall, if successful this study will demonstrate the potential of detecting reduced complexity of HBV DNA integration sites as a biomarker for HBV-HCC screening and provide insight to the involvement of HBV DNA integration sites in HBV-HCC carcinogenesis. Citation Format: Selena Lin, Surbhi Jain, Batbold Boldbaatar, Timothy Block, Wei Song, Ying-Hsiu Su. Comprehensive analysis of the complexity of HBV DNA integration sites in the circulation of patients with HBV-related liver disease. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2014-876

Abstract 888: Development of a urine DNA based marker panel for early detection of liver cancer

Hepatocellular carcinoma (HCC) or liver cancer is an aggressive disease and one of the fastest growing cancers in incidence in the United States. The 5 year survival rate drops dramatically from a poor 26% in early stages to a mere 2% in later stages of the disease. Currently, the only available biomarker for early detection is serum alpha-fetoprotein which can identify only 40-60% of cases. We aim to improve the poor disease prognosis by developing a screening test capable of detecting liver cancer in its earlier stages. Since cancer is a disease of the genome and epigenome, if we can detect these underlying genetic mutations and epigenetic modifications in the periphery, we should be able to effectively detect cancer early. We have previously shown that urine contains fragmented, cell-free, cancer related DNA, both mutated and methylated, derived from the circulation of cancer patients. We have also demonstrated that the concentration of tumor-derived DNA in plasma and in urine is similar in patients with tumors. Hence, we propose the use of a panel of HCC-associated genetic and epigenetic methylation DNA biomarkers to develop a sensitive, noninvasive urine screening test for HCC. In order to detect these markers in the circulation derived urine DNA, we developed short amplicon (∼50 bp) PCR based assays targeting HCC-associated mutations in TP53, CTNNB1, hTERT genes and HCC associated methylation in GSTP1, RASSF1A and CDKN2A. Urine DNA isolated from hepatitis, cirrhosis and HCC patients were tested in each of the assays and analyzed. Panel performance parameters such as sensitivity, specificity and area under the receiver operating curve were calculated for individual markers and then as a group in order to evaluate their ability to distinguish HCC patients from hepatitis and cirrhosis subjects. The potential of this urine test in the early detection of HCC is discussed. Citation Format: Sitong Chen, Surbhi Jain, Selena Lin, Ying-Hsiu Su, Wei Song. Development of a urine DNA based marker panel for early detection of liver cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 888. doi:10.1158/1538-7445.AM2014-888

The stability of herpes simplex virus type I genomes in infected Vero cells undergoing viral induced apoptosis

Maintaining the viral genome intact following infection and prior to replication is critical to the virus life cycle. Here we report an analysis of the stability of herpes simplex virus type 1 (HSV-1) genomes, relative to host chromosomal DNA, in infected cells as a function of viral induced apoptosis. The results show that, in the absence of DNA replication, the input genomes of wild-type (KOS), and replication compromised ICP27 deleted (d27-1) virus are remarkably stable. Intracellular half-lives of their genomes exceeded 24 hours. In contrast, the half-life of replication incompetent ICP4 deleted (d120) viral genomes were significantly less (approximately 8 hours). Interestingly, it was also noted that in cells infected under conditions permissible for replication, viral DNA replication occurs, even in cells undergoing apoptosis. The possibility that the genome structure and replication compartment formation provide protection to the HSV-1 genome from degradation is discussed.

Abstract 5290: ChimericSeq: an easy-to-use program for discovery and analysis of integration events from NGS data

The purpose of this study was to develop a computational method of extracting viral integration events from NGS data in an intuitive way that could accommodate access from users of all disciplines. Viral integration into the host genome is a characteristic of many pathogenic viruses, including the hepatitis B virus (HBV) and human papillomavirus (HPV). Gradual insertion of viral components near proto-oncogenes of the host genome over time can induce uncontrolled cellular proliferation, eventually leading to carcinogenesis. While increased availability of high-throughput next generation sequencing (NGS) has provided tools for researchers to discover these underlying host changes due to viral integrations, there is an emerging need for analytical support of this data. Here we present ChimericSeq, a user-friendly program that can quickly identify viral integration events from NGS data. To fully evaluate this program, we compared the functionality of ChimericSeq to other current viral integration programs. A number of synthetic data sets of HBV sequence fragments integrated into 100bp fragments of random human genomic DNA were created to mimic the nature of chimeric reads of NGS data. ChimericSeq was able to correctly identify reads containing at least 25bp of viral sequence at 100% accuracy. This was a major improvement over the current programs, VirusClip and ViralFusionSeq, which could not detect reads with only 25bp of viral sequence, and even had difficulty correctly identifying viral integration sites containing additional viral sequence. Furthermore, ChimericSeq could detect viral events nearly 10X and 100X faster than these current programs, respectively. Upon testing of NGS data from actual HBV-positive human tissue, we found that ChimericSeq was not only much faster, but was also able to detect more unique viral integration events than current viral integration tools. In conclusion, ChimericSeq expands NGS analytical support to a broader spectrum of the scientific community, being the first program of its kind to offer support in an intuitive graphical user interface (GUI) for commonly used operating platforms, such as Windows and Mac. Citation Format: Patrick Jongeneel, Selena Lin, Jamin Steffen, Surbhi Jain, Ying-Hsiu Su, Wei Song. ChimericSeq: an easy-to-use program for discovery and analysis of integration events from NGS data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5290.

Abstract 1569: Development of a noninvasive and sensitive urine screening test for liver cancer targeting circulation-derived cancer DNA biomarkers

Hepatocellular carcinoma (HCC), or liver cancer, is an aggressive disease and is regarded as one of the fastest growing cancers in the United States in incidence. The 5-year survival rate of HCC patients drops dramatically from a poor 26%, in early-stage cancer, to a mere 2% in late stages of this disease. The only available early detection biomarker is serum alpha-fetoprotein, which can detect only 40-60% of cases. Our goal is to develop a screening test for early detection of liver cancer, in order to meet this urgent need for new and better biomarkers for HCC. Cancer is a disease of the genome and epigenome, and if we can detect these underlying genetic mutations and epigenetic modifications in the periphery, we can effectively detect cancer early. Hence, we chose a panel of both HCC-associated genetic and DNA methylation biomarkers. We have previously shown that urine contains fragmented cell-free DNA derived from the circulation and that we can detect cancer-related DNA, both mutated and methylated DNA, in the urine of such patients. We have also demonstrated that the concentration of tumor-derived DNA in plasma and in urine is similar in patients with tumors. In order to demonstrate the feasibility of detecting HCC DNA biomarkers in the circulation derived urine DNA, we have developed short amplicon (∼50 bp) PCR-based assays targeting HCC-specific mutations in TP53 and CTNNB1 genes and HCC-specific methylation in GSTP1, RASSF1A and CDKN2A genes. Urine DNA isolated from samples of hepatitis, cirrhosis, and HCC patients were tested in each of the assays and analyzed for individual marker and panel performance. The performance of the markers and our approach towards the development of a targeted next generation urine DNA based HCC screening test is discussed. Citation Format: Surbhi Jain, Sitong Chen, Selena Y. Lin, Adam Clemens, Hei-won L. Hann, Ting-Tsung Chang, Chi-Tan Hu, Shun-Hua Chen, Wei Song, Ying-Hsiu Su. Development of a noninvasive and sensitive urine screening test for liver cancer targeting circulation-derived cancer DNA biomarkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1569. doi:10.1158/1538-7445.AM2015-1569